Heparin a polyanionic glycosaminoglycan, remains the most commonly used anticoagulant in patients with acute coronary syndromes and in patients undergoing cardiovascular surgery. While the effects of heparin on the coagulation system have been well documented, abrupt cessation of heparin therapy can lead to a recrudescence of thrombosis and acute ischernia. In addition, clinically unexplained and catastrophic thrombotic complications occur in patients undergoing cardiovascular surgery despite adequate anticoagulation. Endothelial nitric oxide (NO) is an important endogenous inhibitor of platelet-mediated thrombosis; yet, biochemical studies examining the effect of heparin on NO production by the endothelium have heretofore been lacking. In an attempt to address heparin's effect on endothelial cell production of NO, confluent bovine aortic endothelial cells (BAEC) on microcarrier beads were incubated with media containing newborn calf serum and L-arginine, in the presence or absence of heparin. Results indicate that BAEC incubated for with heparin were less able to inhibit platelet aggregation than control cells, and that this effect correlated with a decrease in NO production by BAEC as determined by photolysis-chemiluminescence. A decrease in NO production also occurred after BAEC were exposed to an equimolar concentration of dextran sulfate, suggesting that the decrease in NO after heparin treatment is secondary to its negative charge rather than to a specific saccharide sequence. These data show that heparin, at concentrations achieved in the acute cardiovascular setting, increases platelet aggregation by decreasing endothelial NO production. These observations suggest a mechanism by which to explain, in part, the prothrombotic effects of heparin. The products of the reaction and the mechanism are being investigated by mass spectrometry. Results indicate that the exposure to NO leads to a structural modification of specific sulfated residues. The structural requirements and the modification are being probed with model compounds and stable isotope labeling.